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Liver Microsomes-based In Vitro Metabolism Of T-2Toxin

Posted on:2014-02-22Degree:DoctorType:Dissertation
Country:ChinaCandidate:N N LinFull Text:PDF
GTID:1224330398489938Subject:Drug Analysis
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As an important example, T-2toxin has the most toxicity in presented tens oftrichothecenes mycotoxins. Due to its high toxicity, T-2toxin is considered as apotential chemical agent. It is also one of most dangerous origins for foodcontamination in nature. Currently, residents are greatly treatened by the widepollution of corns and crops caused by T-2toxin. It arouses the related researches onT-2toxin as one of the hot topics. Since T-2toxin metabolizes so rapid, its metabolitesraise the perseverative toxicity in vivo. Therefore, it is especially important todemonstrate the metabolism of T-2toxin.Replacement, reduction and refinement animal testings are the core of the “3R”movement, which is gradually been widely accepted by the international community.Considering that the liver is the main site of drug and toxin metabolism andbioactivation, the in vitro surrogant experimental system based on liver, i.e., the livermicrosomes and hepatocytes, has wide applications for the researches on exogenousmetabolism and relevant toxicology in the“3R”movement with unique advantages andcharacteristics.The aim of this dissertation is to investigate liver microsomes-based in vitrometabolism of T-2toxin, includes the inter-species differences of T-2txoin in livermicrosomes metabolism, especially the metabolic differences in human and severalanimal models, and metabolic effect of different enzyme systems. We expect it canprovide experimental data and scientific evidence for the metabolic mechanism of T-2toxin, the relationship between metabolism and toxicity, and the risk assessment onhuman health and food safety.This dissertation is divided into six chapters. Chapter1is the review ofliteratures, more than70papers are cited. In this chapter recent advances on analysis, in vitro and in vivo metabolisms of T-2toxin are summarized based on the greattoxicological importance of such a toxin.Chapter2describes the preparation of two major metabolites of T-2toxin, HT-2toxin and3’-OH T-2toxin. After separation and purification, both substances arecharacterized by IR,1H NMR and LC-MS techniques. We used in vitro maize esterasetransformation method to convert T-2toxin to HT-2toxin, and the yield was beyond80%. This enzyme catalytic method has the features of site-directed catalysis, mildcondition and high yield. We then adopted in vitro liver S9fraction biotransformationmethod to convert T-2toxin to its hydroxylated product,3’-OH T-2toxin with a yieldhigher than30%. Both reference regents afforded the material base for furtherresearches.On this basis, in Chapter3, we developed several LC-MS/MS methods forsimultaneously quantitative determination of T-2toxin and its major metabolites indifferent matrices, they are, T-2toxin and3’-hydroxy-T-2toxin in rat S9fraction afteradministrated sodium pentobarbital, T-2toxin and its five metabolites in livermicrosomes and rat blood. The methods were fully validated, simple and rapid. Theparameters such as specificity, recovery, matrix effect, precision and accuracy were allmeet the requirements for bioanalysis. Such methods can be further applied in in vitrometabolic reasearches with good potential.Chapter4demonstrates the in vitro metabolism studies of T-2toxin. We foundspecies differences in T-2toxin metabolism in liver microsomes. The descendingorder of intrinsic hepatic clearance rates for T-2toxin was human, mouse, monkey,dog and rat, but the hepatic clearance rates was from mouse, monkey, rat, dog down tohuman. The affinity and enzymatic turnover for T-2toxin were varied in differentanimal species.Chapter5is the in vitro metabolism of T-2in different enzyme systems. We foundthat multiple cytochrome P450(CYP) isoenzyme were involved in T-2toxinmetabolism, including CYP3A4, CYP2E1, CYP1A2, CYP2C9, CYP2B6, CYP2D6and CYP2C19. CYP3A4isoenzyme was the predominant enzyme contributing to T-2 toxin metabolism and the major products were the hydroxylation products. Livermicrosomal esterase played an important role for T-2toxin in liver metabolism, andthe predominant product was HT-2toxin. The contributions originated from differentenzymes were of significant difference for the metabolic rate of T-2toxin. Theesterases played key role in T-2toxin metabolism in human, dog, rat and mousemicrosomes, and CYP450enzymes in monkey microsome.Chaper6we employed electrospray ionization mass spectrometry in thecombination of Q-TOF MS and iontrap MS, and summarized the fragmentation rulesof T-2toxin and its main metabolites. Further, we also demonstrated that there weresix new metabolites related to T-2toxin, and we predicted the structures of suchmetabolites based on previous fragmentation rules.
Keywords/Search Tags:T-2toxin, liver microsomes, metabolites, inter-species difference, cytochrome P450isoenzymes, esterases, metabolism kinetics
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